Pd-ecgf as biomarker of cancer

ABSTRACT

The present invention corresponds to the field of cancer and is related to predicting cancer detection, diagnosis, monitoring and prediction of response to treatment, in particular platelet derived-endothelial cell growth factor (PD-ECGF) levels for their use as a potential value in monitoring disease evolution and predicting response to anti-angiogenic treatment.

FIELD OF THE INVENTION

The present invention belongs to the field of cancer, particularly tobiomarkers useful in cancer diagnosis and prognosis, wherein PD-ECGFbiomarker modified levels in a sample from a subject are useful indiagnosing cancer in a subject or in prognosis of a subject sufferingfrom cancer.

BACKGROUND OF THE INVENTION

The incidence of renal cell carcinoma (RCC) has been steadily risingeach year. There have been significant advances described in ourunderstanding of the biology of this cancer. Thanks to these advances,new therapeutic strategies in patients with advanced disease have beendeveloped.

U.S. Pat. No. 8,029,981B2 relates to methods of diagnosing RCC thatcomprise determining the levels of the hypoxia-inducible protein-2(HIG2). U.S. Pat. No. 7,611,839B2 relates to a method for confirming adiagnosis of RCC in a subject that comprises determining the levels ofelongation factor 1 alpha 2 (EEF1A2) and toll-like receptor 2 (TLR2).

Nevertheless, there are still no biomarkers for routine use in theclinical practice in relation to RCC. Particularly in the last decadethe methods used in prognosis of RCC have not changed. In general thetherapy is classified by the histology; and patients continue to beexposed to potentially toxic therapies with no indication responseprobability. Therefore, there is still a need in the art for identifyingpotential biomarkers in the diagnosis and/or prognosis of RCC.

Worldwide, breast cancer is the second most common type of cancer(10.4%; after lung cancer) and the fifth most common cause of cancerdeath (after lung cancer, stomach cancer, liver cancer, and coloncancer). Among women worldwide, breast cancer is the most common causeof cancer death. The number of cases worldwide has significantlyincreased since the 1970s, a phenomenon partly blamed on modernlifestyles in the Western world. North American women have the highestincidence of breast cancer in the world.

Some well-established breast molecular markers with prognostic and/ortherapeutic value include hormone receptors including estrogen receptor(ER) and progesterone receptor (PR), HER-2 oncogene, Ki-67, and p53.More recently identified molecular targets in breast cancer includeCXCR4, caveolin and FOXP3.

With 655,000 deaths worldwide per year, colorectal cancer is the thirdmost common form of cancer and the second leading cause ofcancer-related death in the Western world. Many colorectal cancers arethought to arise from adenomatous polyps in the colon. Thesemushroom-shaped growths are usually benign, but some may develop intocancer over time. The majority of the time, the diagnosis of localizedcolon cancer is through colonoscopy.

Surgical resection is the primary treatment modality for early stagecolorectal cancer (stage I through III), and the most powerful tool forassessing prognosis following potentially curative surgery is pathologicanalysis of the resected specimen. Although the parameters thatdetermine pathologic stage are the strongest predictors of postoperativeoutcome, other clinical, molecular, and histologic features mayinfluence prognosis independent of stage. Among patients with stage IVdisease, prognosis is more closely tied to the location and extent ofdistant metastatic disease

Carcinoembryonic antigen (CEA) level is the tumor marker most often usedin colorectal cancer. This level can be checked prior to surgery topredict prognosis, can be used during therapy to assess response totreatment or after completion of therapy to monitor for recurrence. CA19-9 is a blood marker that may be elevated in colorectal cancer. MSI(microsatellite instability) can be used to identify early stage coloncancer that may require more aggressive treatment or to identifypatients who should have further genetic testing due to the risk for afamilial syndrome related to several cancer types. Recently, serummiR-21 has been proposed as an early diagnostic and prognostic biomarkerin colorectal cancer (Toiyama et al. 2013 J. Natl. Cancer Inst. 105(12):849-859).

However, due to the high incidence of both breast cancer and colorectalcancer, there is a continuing need in the art for diagnostic and/orpredictive markers of response of said cancers.

SUMMARY OF THE INVENTION

The inventors have analyzed expression levels of PD-ECGF protein byinmunohistochemistry, ELISA and western blotting. PD-ECGF was notdetected in non-cancerous tissue samples (0 in 12, 0%), but PD-ECGFprotein was observed in 67 (>97%) tissue samples out of 69 RCC patientsand in 10 (59%) out of 17 in tissue samples from breast cancer patientsanalyzed by inmunohistochemistry. Thus, PD-ECGF determination fromtissue samples could be used for cancer detection, diagnosis ormonitoring.

In addition, the inventors have found that PD-ECGF expression is apredictive factor of the response to anti-angiogenic therapies inpatients with renal cell carcinoma, and colorectal cancer as shown bycorrelating PD-ECGF levels in the tumors prior to the initiation of thetherapy with the risk of early progression (Kaplan-Meier andGehan-Breslow-Wilcoxon tests, p<0.045). Therefore, PD-ECGF expressionhas potential value in predicting cancer progression to therapy as apredictive factor of response to anti-angiogenic therapies.

Furthermore, colorectal patients with higher plasma levels of PD-ECGFhad a significantly higher risk of early progression, as represented bya significant hazard ratio analysis for PD-ECGF and tumor progression(Hazard ratio, p<0.041). Therefore, PD-ECGF plasma protein levels have apotential value in predicting cancer response to therapy as a predictivefactor of response to anti-angiogenic therapies.

These findings support the use of PD-ECGF as a novel biomarker fordetection, diagnosis, disease monitoring and prediction of response inpatients with RCC, breast cancer, colorectal cancer and other type ofcancers.

Thus, in a first aspect, the present invention relates to a method forthe diagnosis of cancer in a subject that comprises

(i) determining the levels of PD-ECGF in a sample from said subject and,

(ii) comparing the levels obtained in (i) to a reference value,

wherein

-   -   if the levels of PD-ECGF in a sample from the subject are        increased when compared to a reference value, then the subject        is diagnosed with cancer, and    -   if the levels of PD-ECGF in a sample from the subject are        decreased when compared to a reference value, then the subject        is not diagnosed with cancer.

In another aspect, the invention relates to a method for predicting theresponse of a subject suffering from cancer to an anti-angiogenictreatment, wherein said anti-angiogenic treatment is not doxorubicin orinterferon therapy, that comprises

(i) determining the levels of PD-ECGF in a sample from said subject and,

(ii) comparing the levels obtained in (i) to a reference value,

wherein

-   -   if the levels of PD-ECGF in a sample from the subject are        increased when compared to a reference value, it is indicative        of a bad response to the anti-angiogenic treatment, and    -   if the levels of PD-ECGF in a sample from the subject are        decreased when compared to a reference value, it is indicative        of a good response to the anti-angiogenic treatment.

In a further aspect, the invention relates to the use of PD-ECGF in thediagnosis of cancer in a subject and/or in the determination of theresponse of a cancer patient to an anti-angiogenic treatment, whereinsaid anti-angiogenic treatment is not doxorubicin or interferon therapy.

DESCRIPTION OF THE FIGURES

FIG. 1. Levels of PD-ECGF protein from tumor or non-tumor tissue samplesof patients or healthy subjects measured by inmunohistochemistry.

FIG. 2. Levels of PD-ECGF protein from tumor tissue samples of RCCCancer patients measured by inmunohistochemistry.

FIG. 3. Levels of PD-ECGF protein from tumor tissue samples of BreastCancer patients measured by inmunohistochemistry.

FIG. 4. Kaplan-Meier survival plot of progression free survival in RCCpatients related with the tumor tissue levels of PD-ECGF expression.

FIG. 5. PD-ECGF protein levels measured by ELISA from plasma samples ofhealthy donors or cancer patients.

FIG. 6. PD-ECGF protein levels measured by ELISA from plasma samples ofRCC cancer patients.

FIG. 7. PD-ECGF protein levels measured by ELISA from plasma samples ofBreast Cancer patients.

FIG. 8. PD-ECGF protein levels measured by ELISA from plasma samples ofcolorectal cancer patients.

FIG. 9. Hazard Ratio association of PD-ECGF plasma protein levels totumor progression in colorectal cancer patients.

FIG. 10. Kaplan-Meier survival plot of progression free survival in RCCpatients related with the plasma levels of PD-ECGF.

FIG. 11. Kaplan-Meier disease-free survival plot of colorectaladenocarcinoma patients wherein PD-ECGF expression is altered. Allcomplete tumors (7 samples). Reference: Colorectal Adenocarcinoma (TCGA,Provisional).

FIG. 12. Kaplan-Meier overall survival plot of glioblastoma patientswherein PD-ECGF expression is altered. All complete tumors (291samples). Reference: Brennan C W et al., The somatic genomic landscapeof glioblastoma. Cell. 2013 Oct. 10; 155(2):462-77.

FIG. 13. Kaplan-Meier overall survival plot of kidney renal clear cellcarcinoma patients wherein PD-ECGF expression is altered. All completetumors (413 samples). Reference: Kidney Renal Clear Cell Carcinoma(TCGA, Provisional).

FIG. 14. Kaplan-Meier disease-free survival plot of kidney renal clearcell carcinoma patients wherein PD-ECGF expression is altered. Allcomplete tumors (413 samples). Reference: Kidney Renal Clear CellCarcinoma (TCGA, Provisional).

FIG. 15. Kaplan-Meier overall survival plot of kidney renal clear cellcarcinoma patients wherein PD-ECGF expression is altered. All completetumors (392 samples). Reference: Cancer Genome Atlas Research Network.Comprehensive molecular characterization of clear cell renal cellcarcinoma. Nature. 2013 Jul. 4; 499(7456):43-9.

FIG. 16. Kaplan-Meier disease-free survival plot of kidney renalpapillary cell carcinoma patients wherein PD-ECGF expression is altered.All complete tumors (161 samples). Reference: Kidney Renal PapillaryCell Carcinoma (TCGA, Provisional).

FIG. 17. Kaplan-Meier overall survival plot of lung adenocarcinomapatients wherein PD-ECGF expression is altered. All complete tumors (230samples). Reference: Lung Adenocarcinoma (TCGA, Provisional).

FIG. 18. Kaplan-Meier disease-free survival plot of lung adenocarcinomapatients wherein PD-ECGF expression is altered. All complete tumors (230samples). Reference: Lung Adenocarcinoma (TCGA, Provisional).

FIG. 19. Kaplan-Meier disease-free survival plot of prostateadenocarcinoma patients wherein PD-ECGF expression is altered. Allcomplete tumors (85 samples). Reference: Taylor B S et al., Integrativegenomic profiling of human prostate cancer. Cancer Cell. 2010 Jul. 13;18(1):11-22.

FIG. 20. Kaplan-Meier overall survival plot of testicular germ cellcancer patients wherein PD-ECGF expression is altered. All completetumors (149 samples). Reference: Testicular Germ Cell Cancer (TCGA,Provisional).

FIG. 21. Kaplan-Meier overall survival plot of thymoma patients whereinPD-ECGF expression is altered. All complete tumors (124 samples).Reference: Thymoma (TCGA, Provisional).

FIG. 22. Kaplan-Meier disease-free survival plot of thymoma patientswherein PD-ECGF expression is altered. All complete tumors (124samples). Reference: Thymoma (TCGA, Provisional).

FIG. 23. Kaplan-Meier overall survival plot of papillary thyroidcarcinoma patients wherein PD-ECGF expression is altered. All completetumors (388 samples). Reference: Cancer Genome Atlas Research Network.Integrated genomic characterization of papillary thyroid carcinoma.Cell. 2014 Oct. 23; 159(3):676-90.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “anti-angiogenic treatment” or “anti-angiogenesis treatment”,as used herein, relates to a treatment based on at least oneanti-angiogenesis agent. The term “anti-angiogenic agent” or“anti-angiogenesis agent” or “angiogenesis inhibitor”, relates to anagent targeted to angiogenesis (e.g. the process of forming bloodvessels) including, but not limited to, tumor angiogenesis. In thiscontext, inhibition can refer to blocking the formation of blood vesselsand halting or slowing down the growth of blood vessels.

The term “cancer” is referred to a disease characterized by uncontrolledcell division (or by an increase of survival or apoptosis resistance)and by the ability of said cells to invade other neighboring tissues(invasion) and spread to other areas of the body where the cells are notnormally located (metastasis) through the lymphatic and blood vessels,circulate through the bloodstream, and then invade normal tissueselsewhere in the body. Depending on whether or not they can spread byinvasion and metastasis, tumors are classified as being either benign ormalignant: benign tumors are tumors that cannot spread by invasion ormetastasis, i.e., they only grow locally; whereas malignant tumors aretumors that are capable of spreading by invasion and metastasis.Biological processes known to be related to cancer include angiogenesis,immune cell infiltration, cell migration and metastasis. The term cancerincludes, without limitation, lung cancer, sarcoma, malignant melanoma,pleural mesothelioma, bladder carcinoma, prostate cancer, pancreascarcinoma, gastric carcinoma, ovarian cancer, hepatoma, breast cancer,colorectal cancer, kidney cancer, esophageal cancer, suprarenal cancer,parotid gland cancer, head and neck carcinoma, cervix cancer,endometrial cancer, liver cancer, mesothelioma, multiple myeloma,leukaemia, and lymphoma. In a particular embodiment of the invention,the cancer is renal cell carcinoma (RCC), breast cancer, or colorectalcancer. The term “breast cancer” relates to any malignant proliferativedisorder of breast cells, most commonly from the inner lining of milkducts or the lobules that supply the ducts with milk. Cancersoriginating from ducts are known as ductal carcinomas, while thoseoriginating from lobules are known as lobular carcinomas. The term“colorectal cancer” also known as “colon cancer”, “rectal cancer”, or“bowel cancer”, refers to a cancer from uncontrolled cell growth in thecolon or rectum, or in the appendix. The term “renal cell carcinoma”also known as “kidney cancer” or “renal adenocarcinoma” relates tocancer wherein tumor cells are found in any tissue of the kidneyincluding clear cell carcinomas (mixed with granular cells or not),chromophilic cancers, rhabdoid tumors of the kidney, chromophobiccancers, oncocytic cancers, collecting duct cancers, transitional cellcarcinomas and sarcomatoid tumors.

The term “diagnosis”, as used herein, refers both to the process ofattempting to determine and/or identify a possible disease in a subject,i.e. the diagnostic procedure, and to the opinion reached by thisprocess, i.e. the diagnostic opinion. As such, it can also be regardedas an attempt at classification of an individual's condition intoseparate and distinct categories that allow medical decisions abouttreatment and prognosis to be made. In particular, the term “diagnosisof cancer” relates to the capacity to identify or detect the presence ofa tumor in a subject. This detection, as it is understood by a personskilled in the art, does not claim to be correct in 100% of the analyzedsamples. However, it requires that a statistically significant amount ofthe analyzed samples are classified correctly. The amount that isstatistically significant can be established by a person skilled in theart by means of using different statistical tools; illustrative,non-limiting examples of said statistical tools include determiningconfidence intervals, determining the p-value, the Student's t-test orFisher's discriminant functions, etc. (see, for example, Dowdy andWearden, Statistics for Research, John Wiley & Sons, New York 1983). Theconfidence intervals are preferably at least 90%, at least 95%, at least97%, at least 98% or at least 99%. The p-value is preferably less than0.1, less than 0.05, less than 0.01, less than 0.005 or less than0.0001. The teachings of the present invention preferably allowcorrectly diagnosing in at least 60%, in at least 70%, in at least 80%,or in at least 90% of the subjects of a determined group or populationanalyzed.

The term “expression level”, as used herein, refers to the measurablequantity of gene product produced by the gene in a sample of thesubject, wherein the gene product can be a transcriptional product or atranslational product. As understood by the person skilled in the art,the gene expression level can be quantified by measuring the messengerRNA levels of said gene or of the protein encoded by said gene. In thecontext of the present invention, the expression level of the geneencoding PD-ECGF can be determined by measuring the levels of mRNAencoded by said gene, or by measuring the levels of the protein encodedby said gene, i.e. PD-ECGF protein or of variants thereof. PD-ECGFprotein variants include all the physiologically relevantpost-translational chemical modifications forms of the protein, forexample, glycosylation, phosphorylation, acetylation, etc., providedthat the functionality of the protein is maintained. Said termencompasses the PD-ECGF protein of any mammal species, including but notbeing limited to domestic and farm animals (cows, horses, pigs, sheep,goats, dogs, cats or rodents), primates and humans. Preferably, thePD-ECGF protein is a human protein.

The term “plateled derived-endothelial cell growth factor” or “PD-ECGF”,as used herein, is also known as “plateled-derived endothelial cellgrowth factor-1”, “ECGF-1” or “ECGF1”, “gliostatin”, “platelet-derivedendothelial cell mitogen” “thymidine phosphorylase” or “TP”, and relatesto a cytoplasmic protein initially isolated from platelets showingendothelial mitogenic activity. It is an acidic non-glycosylated proteinof 45 kDa, which is synthesized as a precursor of 482 amino acids fromwhich it is derived by N-terminal processing. The protein isolated fromplacenta contains five additional amino acids at the N-terminus. PD-ECGFcan be phosphorylated in vivo at serine residues but the biologicalsignificance of this phosphorylation step is unknown. The human geneencoding PD-ECGF is located on chromosome 22 and assigned Gene ID 1890(NCBI GenBank, 1 Mar. 2014 update). A number of transcript variants havebeen described for PD-ECGF: transcript variant 1 (accession number NM001113755.2 in NCBI GenBank), encoding the same isoform 1 than variants2, 3 and 4; transcript variant 2 (NM 001953.4), which uses an alternatesplice site in the 5′ UTR; transcript variant 3 (NM 001113756.2), whichdiffers in the 5′ UTR compared to variant 1; transcript variant 4 (NM001257988.1), which uses an alternate splice site in the 5′ UTR comparedto variant 1; and transcript variant 5 (NM_001257989.1), which usesalternate splice sites in the 5′ UTR and the 3′ coding region comparedto variant 1, and coding for isoform 2, said isoform 2 having anadditional segment in the C-terminal region compared to isoform 1. Theamino acid sequence of human PD-ECGF is located in NCBI GenBank underaccession number AAB03344.2 (482 amino acids, version as of 3 Feb. 2000)and under UniProtKB/Swiss-Prot accession number P19971.2 (Uniprotversion 167 as of 19 Feb. 2014).

The term “predicting the response to an anti-angiogenic treatment”, asused herein, relates to the prediction of a medical outcome following atherapeutic intervention using an anti-angiogenic treatment. The outcomeafter the treatment may be determined using any common end point forpatient progression, such as, for example, a poor or good outcome (e.g.,likelihood of long-term survival, overall survival, disease-specificsurvival, progression-free survival or disease-free survival); relapse,disease progression, or mortality. As will be understood by thoseskilled in the art, the prediction of the response, although preferredto be, need not be correct for 100% of the subjects to be diagnosed orevaluated. The term, however, requires that a statistically significantportion of subjects can be identified as having an increased probabilityof having a given outcome in response to the therapy. Whether a subjectis statistically significant can be determined without further ado bythe person skilled in the art using various well known statisticevaluation tools, e.g., determination of confidence intervals, p-valuedetermination, Student's t-test, Mann-Whitney test, etc. Details arefound in Dowdy and Wearden, Statistics for Research, John Wiley & Sons,New York 1983. Preferred confidence intervals are at least 50%, at least60%, at least 70%, at least 80%, at least 90% at least 95%. The p-valuesare, preferably 0.05, 0.025, 0.001 or lower. Any parameter which iswidely accepted for determining response of a patient can be used in thepresent invention including, without limitation of disease progression.

The term “reference value”, as used herein, refers to a laboratory valueused as a reference for values/data obtained by laboratory examinationsof subjects or samples collected from subjects. The reference value orreference level can be an absolute value, a relative value, a value thathas an upper or a lower limit, a range of values, an average value, amedian value, a mean value, or a value as compared to a particularcontrol or baseline value. A reference value can be based on anindividual sample value, such as for example, a value obtained from asample from the subject being tested, but at an earlier point in time.The reference value can be based on a large number of samples, such asfrom population of subjects of the chronological age matched group, orbased on a pool of samples including or excluding the sample to betested. Suitable reference values are indicated in the context of themethods of the invention for determining cancer diagnosis or predictingthe response of a subject with cancer that is being treated withanti-angiogenic therapies.

The term “sample” or “biological sample”, as used herein, refers tobiological material isolated from a subject. The biological samplecontains any biological material suitable for detecting RNA or proteinlevels. In a particular embodiment, the sample comprises geneticmaterial, e.g., DNA, genomic DNA (gDNA), complementary DNA (cDNA), RNA,heterogeneous nuclear RNA (hnRNA), mRNA, etc., from the subject understudy. The sample can be isolated from any suitable tissue or biologicalfluid such as, for example blood, saliva, plasma, serum, urine,cerebrospinal liquid (CSF), feces, a surgical specimen, a specimenobtained from a biopsy, and a tissue sample embedded in paraffin.Methods for isolating samples are well known to those skilled in theart. In particular, methods for obtaining a sample from a biopsy includegross apportioning of a mass, or micro-dissection or other art-knowncell-separation methods. In order to simplify conservation and handlingof the samples, these can be formalin-fixed and paraffin-embedded orfirst frozen and then embedded in a cryosolidifiable medium, such asOCT-Compound, through immersion in a highly cryogenic medium that allowsrapid freeze. In a particular embodiment, the sample from the subjectaccording to the methods of the present invention is a biological fluidsample. In a particular embodiment, the sample from the subjectaccording to the methods of the present invention is selected from thegroup consisting of blood, serum, plasma, and a tissue sample; morepreferably from the group consisting of plasma and a tissue sample.

The term “subject” or “individual” or “animal” or “patient” or “mammal,”relates to all the animals classified as mammals and includes but is notlimited to domestic and farm animals, primates and humans, for example,human beings, non-human primates, cows, horses, pigs, sheep, goats,dogs, cats, or rodents. Preferably, the subject is a male or femalehuman being of any age, sex or race.

The term “treatment”, as used herein comprises any type of therapy,which aims at terminating, preventing, ameliorating and/or reducing thesusceptibility to a clinical condition as described herein. In apreferred embodiment, the term treatment relates to prophylactictreatment (i.e. a therapy to reduce the susceptibility of a clinicalcondition, a disorder or condition as defined herein). Thus,“treatment,” “treating,” and the like, as used herein, refer toobtaining a desired pharmacologic and/or physiologic effect, coveringany treatment of a pathological condition or disorder in a mammal,including a human. The effect may be prophylactic in terms of completelyor partially preventing a disorder or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disorder and/oradverse effect attributable to the disorder. That is, “treatment”includes (1) preventing the disorder from occurring or recurring in asubject, (2) inhibiting the disorder, such as arresting its development,(3) stopping or terminating the disorder or at least symptoms associatedtherewith, so that the host no longer suffers from the disorder or itssymptoms, such as causing regression of the disorder or its symptoms,for example, by restoring or repairing a lost, missing or defectivefunction, or stimulating an inefficient process, or (4) relieving,alleviating, or ameliorating the disorder, or symptoms associatedtherewith, where ameliorating is used in a broad sense to refer to atleast a reduction in the magnitude of a parameter, such as inflammation,pain, and/or immune deficiency.

Cancer Diagnostic Method of the Invention

The authors of the present invention have found that PD-ECGF expressionis not detected in non-cancerous tissue samples (0 in 12, 0%), whilePD-ECGF protein expression is detected in 67 (>97%) tissue samples outof 69 RCC patients and in 9 (56%) out of 16 in tissue samples frombreast cancer patients when tissue samples were analyzed byimmunohistochemistry (see Example 1). Similarly, PD-ECGF expression wasobserved in 14 (>87%) plasma samples out of 16 RCC patients, in 11 (69%)out of 16 in plasma samples from breast cancer patients and in 52 (90%)out of 58 in plasma samples from colorectal cancer patients analyzed byELISA (see Example 2).

Therefore, in a first aspect, the invention relates to a method for thediagnosis of cancer in a subject that comprises

(i) determining the levels of PD-ECGF in a sample from said subject and,

(ii) comparing the levels obtained in (i) to a reference value,

wherein

-   -   if the levels of PD-ECGF in a sample from the subject are        increased when compared to a reference value, then the subject        is diagnosed with cancer, and    -   if the levels of PD-ECGF in a sample from the subject are        decreased when compared to a reference value, then the subject        is not diagnosed with cancer.

Thus, in a first step of the diagnostic method of the invention, theexpression levels of PD-ECGF are determined in a sample from the subjectwhose diagnosis is to be determined. The sample wherein the expressionlevel of PD-ECGF is determined can be any sample containing cells fromthe potential tumor. In a particular embodiment, the sample containingcells from the potential tumor is a biological fluid sample. In aparticular embodiment, the sample containing cells from the potentialtumor is potential tumor tissue or a portion thereof. In a moreparticular embodiment, said potential tumor tissue sample is a kidneytissue sample from a patient whose diagnosis of kidney cancer is to bedetermined, or a breast tissue sample from a patient whose diagnosis ofbreast cancer is to be determined, or a colorectal tissue sample from apatient whose diagnosis of colorectal cancer is to be determined. Saidsample can be obtained by conventional methods, e.g., biopsy, surgicalexcision or aspiration, by using methods well known to those of ordinaryskill in the related medical arts. Methods for obtaining the sample fromthe biopsy include gross apportioning of a mass, or microdissection orother art-known cell-separation methods including nephrectomy andpartial tumorectomy. Tumor cells can additionally be obtained from fineneedle aspiration cytology. In order to simplify conservation andhandling of the samples, these can be formalin-fixed andparaffin-embedded or first frozen and then embedded in acryosolidifiable medium, such as OCT-Compound, through immersion in ahighly cryogenic medium that allows for rapid freeze.

In another embodiment, the sample wherein the expression level ofPD-ECGF is determined is a biofluid from the patient whose diagnosis isto be determined. In a preferred embodiment, the biofluid is selectedfrom blood, particularly peripheral blood, plasma or serum. The bloodsample is typically extracted by means of puncturing an artery or vein,normally a vein from the inner part of the elbow or from the back of thehand, the blood sample being collected in an air-tight vial or syringe.A capillary puncture normally on the heel or on the distal phalanxes offingers can be performed for analysis by means of a micromethod. Serumcan be obtained from the complete blood sample and in the absence ofanticoagulant by leaving the sample to settle for 10 minutes so that itcoagulates and subsequently centrifuging it at 1,500 rpm for 10 minutesfor the purpose of separating the cells (precipitate) from the serum(supernatant). In turn, to obtain the plasma sample the complete bloodis contacted with an anticoagulant and is centrifuged at 3,000 rpm for20 minutes. The precipitate of said centrifugation corresponds to theformed elements, and the supernatant corresponds to the plasma. Theserum or the plasma obtained can be transferred to a storage tube forsample analysis by means of the method of the invention.

In a particular embodiment of the diagnostic method of the invention,the sample wherein PD-ECGF expression levels are determined is a plasmasample or a tissue sample. In a particular embodiment, PD-ECGFexpression levels are determined in a plasma sample or in a breasttissue sample when diagnosis of breast cancer is to be determined. In aparticular alternative embodiment, PD-ECGF expression levels aredetermined in a plasma sample or in a kidney tissue sample whendiagnosis of renal cell carcinoma (RCC) is to be determined. In anotherparticular alternative embodiment, PD-ECGF expression levels aredetermined in a plasma sample or in a colorectal tissue sample whendiagnosis of colorectal cancer is to be determined.

As previously described, gene expression levels can be quantified bymeasuring the messenger RNA levels of the gene or of the protein encodedby said gene or of the protein encoded by said gene, i.e. PD-ECGFprotein or of variants thereof. PD-ECGF protein variants include all thephysiologically relevant post-translational chemical modifications formsof the protein, for example, glycosylation, phosphorylation,acetylation, etc., provided that the functionality of the protein ismaintained. Said term encompasses the PD-ECGF protein of any mammalspecies, including but not being limited to domestic and farm animals(cows, horses, pigs, sheep, goats, dogs, cats or rodents), primates andhumans. Preferably, the PD-ECGF protein is a human protein.

In order to measure the mRNA levels of a gene, the biological sample maybe treated to physically, mechanically or chemically disrupt tissue orcell structure, to release intracellular components into an aqueous ororganic solution to prepare nucleic acids for further analysis. Thenucleic acids are extracted from the sample by procedures known to theskilled person and commercially available. RNA is then extracted fromfrozen or fresh samples by any of the methods typical in the art, forexample, Sambrook, J., et al., 2001. Molecular cloning: A LaboratoryManual, 3^(rd) ed., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3.Preferably, care is taken to avoid degradation of the RNA during theextraction process.

The expression level can be determined using mRNA obtained from aformalin-fixed, paraffin-embedded tissue sample. mRNA may be isolatedfrom an archival pathological sample or biopsy sample which is firstdeparaffinized. An exemplary deparaffinization method involves washingthe paraffinized sample with an organic solvent, such as xylene.Deparaffinized samples can be rehydrated with an aqueous solution of alower alcohol. Suitable lower alcohols, for example, include methanol,ethanol, propanols and butanols. Deparaffinized samples may berehydrated with successive washes with lower alcoholic solutions ofdecreasing concentration, for example. Alternatively, the sample issimultaneously deparaffinized and rehydrated. The sample is then lysedand RNA is extracted from the sample. Samples can be also obtained fromfresh tumor tissue such as a resected tumor. In a particular embodimentsamples can be obtained from fresh tumor tissue or from OCT embeddedfrozen tissue. In another preferred embodiment samples can be obtainedby colonoscopy and then paraffin-embedded.

In order to normalize the values of mRNA expression among the differentsamples, it is possible to compare the expression levels of the mRNA ofinterest in the test samples with the expression of a control RNA. A“control RNA” as used herein, relates to RNA whose expression levels donot change or change only in limited amounts in tumor cells with respectto non-tumorigenic cells. Preferably, the control RNA is mRNA derivedfrom housekeeping genes and which code for proteins which areconstitutively expressed and carry out essential cellular functions.Preferred housekeeping genes for use in the present invention includeβ-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, IPO8,HPRT, GAPDH, PSMB4, tubulin and β-actin. In a preferred embodiment, thecontrol RNA is GAPDH, IPO8, HPRT, β-actin, 18-S ribosomal protein orPSMB4 mRNA.

In one embodiment relative gene expression quantification is calculatedaccording to the comparative threshold cycle (Ct) method using GAPDH,IPO8, HPRT, β-actin or PSMB4 as an endogenous control and commercial RNAcontrols as calibrators. Final results are determined according to theformula 2-(ΔCt sample-ΔCt calibrator), where ΔCT values of thecalibrator and sample are determined by subtracting the Ct value of thetarget gene from the value of the control gene.

Suitable methods to determine gene expression levels at the mRNA levelinclude, without limitation, standard assays for determining mRNAexpression levels such as qPCR, RT-PCR, RNA protection analysis,Northern blot, RNA dot blot, in situ hybridization, microarraytechnology, tag based methods such as serial analysis of gene expression(SAGE) including variants such as LongSAGE and SuperSAGE, microarrays,fluorescence in situ hybridization (FISH), including variants such asFlow-FISH, qFiSH and double fusion FISH (D-FISH), and the like.

Suitable methods to determine gene expression levels at the proteinlevel include, without limitation, conventional methods for determiningprotein expression levels, such as using antibodies with a capacity tospecifically bind to the proteins encoded by said genes (or to fragmentsthereof containing antigenic determinants) and subsequent quantificationof the resulting antibody-antigen complexes. In a particular embodiment,PD-ECGF protein levels can be quantified by using standard assays fordetermining protein expression levels such as Western-blot or Westerntransfer, ELISA (enzyme-linked immunosorbent assay), RIA(radioimmunoassay), competitive EIA (competitive enzyme immunoassay),DAS-ELISA (double antibody sandwich ELISA), immunocytochemical andimmunohistochemical techniques, techniques based on the use of proteinbiochips or microarrays which include specific antibodies or assaysbased on colloidal precipitation in formats such as dipsticks.

The antibodies to be employed in these assays can be, for example,polyclonal sera, hybridoma supernatants or monoclonal antibodies,antibody fragments, Fv, Fab, Fab′ and F(ab′)2, ScFv, diabodies,triabodies, tetrabodies and humanized antibodies. At the same time, theantibodies can be labeled or not. Illustrative, but non-exclusiveexamples of markers which can be used include radioactive isotopes,enzymes, fluorophores, chemiluminescent reagents, enzymatic substratesor cofactors, enzymatic inhibitors, particles, colorants, etc. There area wide variety of well-known assays that can be used in the presentinvention, which use non-labeled antibodies (primary antibody) andlabeled antibodies (secondary antibodies); among these techniques areincluded Western blot or Western transfer, ELISA, RIA, competitive EIA,DAS-ELISA, immunocytochemical and immunohistochemical techniques,techniques based on the use of biochips or protein microarrays includingspecific antibodies or assays based on colloidal precipitation informats such as dipsticks. Other ways of detecting and quantifying thelevels of the protein of interest include techniques of affinitychromatography, binding-ligand assays, etc.

On the other hand, the determination of the levels of the PD-ECGFprotein can be carried out by constructing a tissue microarray (TMA)containing the subject samples assembled, and determining the expressionlevels of the corresponding protein by immunohistochemistry techniques.Immunostaining intensity can be evaluated by two or more differentpathologists and scored using uniform and clear cut-off criteria, inorder to maintain the reproducibility of the method. Discrepancies canbe resolved by simultaneous re-evaluation. Briefly, the result ofimmunostaining can be recorded as negative expression (0) versuspositive expression, and low expression (1+) versus moderate (2+) andhigh (3+) expression, taking into account the expression in tumor cellsand the specific cut-off for each marker. As a general criterion, thecut-offs are selected in order to facilitate reproducibility, and whenpossible, to translate biological events. Alternatively, theimmunostaining intensity can be evaluated by using imaging techniquesand automated methods such as those disclosed in Rojo, M. G. et al.(Folia Histochem. Cytobiol. 2009; 47: 349-54) or Mulrane, L. et al.(Expert Rev. Mol. Diagn. 2008; 8: 707-25).

Alternatively, in another particular embodiment, the levels of thePD-ECGF protein are determined by Western blot. Western blot is based onthe detection of proteins previously resolved by gel electrophoresesunder denaturing conditions and immobilized on a membrane, generallynitrocellulose, by the incubation with an antibody specific and adeveloping system (e.g. chemoluminiscent).

In a particular embodiment, PD-ECGF expression levels to be determinedin the diagnostic method of the invention are determined as PD-ECGFprotein levels. In a more particular embodiment, PD-ECGF protein levelsare determined by ELISA, western blot or by immunohistochemistry.

The term “activity level” of a protein, more particularly of an enzyme,as used herein refers to a measure of the enzyme activity, particularlymeasured as moles of substrate converted per unit of time.

Assays to determine the activity level of an enzyme are known by theskilled person and include, without limitation, initial rate assays,progress curve assays, transient kinetics assays and relaxation assays.Continuous assays of enzymatic activity include, without limitation,spectrophotometric, fluorometric, calorimetric, chemiluminiscent, lightscattering and microscale thermopheresis assays. Discontinuous assays ofenzymatic activity include, without limitation, radiometric andchromatographic assays. As the skilled person understands, factors thatmay influence enzymatic activity comprise salt concentration,temperature, pH, and substrate concentration.

In a second step of the diagnostic method of the invention, theexpression levels of PD-ECGF in the sample from a subject whosediagnosis is to be determined are compared to a reference value.

In the context of the method of the invention for the diagnosis ofcancer in a subject, the reference value is the PD-ECGF expression leveldetermined in a sample from a healthy subject, i.e. a subject notdiagnosed with cancer, or in a non-tumor tissue sample from a subjectdiagnosed with cancer, preferably the reference value is the PD-ECGFexpression level determined a sample from a healthy subject, or in asubject not diagnosed with cancer.

Once this reference value is established, the level of PD-ECGF expressedin the sample can be compared with said reference value, and thus beassigned a level of “increased” or “decreased” expression. For example,an increase in expression levels above the reference value of at least1.1-fold, 1.5-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more compared withthe reference value is considered as “increased” expression level. Onthe other hand, a decrease in expression levels below the referencevalue of at least 0.9-fold, 0.75-fold, 0.2-fold, 0.1-fold, 0.05-fold,0.025-fold, 0.02-fold, 0.01-fold, 0.005-fold or even less compared withreference value is considered as “decreased” expression level.

Thus, if an increased expression level of PD-ECGF in the sample from asubject whose diagnosis is to be determined when compared to a referencevalue is observed, then the subject is diagnosed with cancer.Alternatively, if a decreased expression level of PD-ECGF in the samplefrom a subject whose diagnosis is to be determined when compared to thereference value, then the subject is not diagnosed with cancer.

Method for Predicting the Response to an Anti-Angiogenic TreatmentAccording to the Invention

The authors of the present invention have found that the levels ofPD-ECGF determined in cancer patients show a statistically significantcorrelation with the risk that the patients shows early progressionafter treatment with an anti-angiogenic therapy. Thus, in a furtheraspect, the invention relates to a method for predicting the response totreatment in a subject suffering from cancer and undergoing ananti-tumoral treatment that comprises

(i) determining the levels of PD-ECGF in a sample from said subject and,

(ii) comparing the levels obtained in (i) to a reference value,

wherein

-   -   if the levels of PD-ECGF in a sample from the subject are        increased when compared to a reference value, it is indicative        of a poor response of the cancer patient to the treatment, and    -   if the levels of PD-ECGF in a sample from the subject are        decreased when compared to a reference value, it is indicative        of a good response of the cancer patient to the treatment.

In a particular embodiment, the invention relates to a method forpredicting the response of a subject suffering from cancer to ananti-angiogenic treatment that comprises

(i) determining the levels of PD-ECGF in a sample from said subject and,

(ii) comparing the levels obtained in (i) to a reference value,

wherein

-   -   if the levels of PD-ECGF in a sample from the subject are        increased when compared to a reference value, it is indicative        of a bad response to the anti-angiogenic treatment, and    -   if the levels of PD-ECGF in a sample from the subject are        decreased when compared to a reference value, it is indicative        of a good response to the anti-angiogenic treatment.

In a more particular embodiment, the invention relates to a method forpredicting the response of a subject suffering from cancer to ananti-angiogenic treatment, wherein said anti-angiogenic treatment is notdoxorubicin or interferon therapy, that comprises

(i) determining the levels of PD-ECGF in a sample from said subject and,

(ii) comparing the levels obtained in (i) to a reference value,

wherein

-   -   if the levels of PD-ECGF in a sample from the subject are        increased when compared to a reference value, it is indicative        of a bad response to the anti-angiogenic treatment, and    -   if the levels of PD-ECGF in a sample from the subject are        decreased when compared to a reference value, it is indicative        of a good response to the anti-angiogenic treatment.

In a first step of the method of the invention for predicting theresponse to an anti-angiogenic treatment, the expression levels ofPD-ECGF are determined in a sample from a subject suffering from cancerwhose response to treatment is to be predicted.

The sample wherein the expression level of PD-ECGF is determined can beany sample containing cells from the tumor. In a particular embodiment,the sample containing cells from the potential tumor is a biologicalfluid sample. In a particular embodiment, the sample containing cellsfrom the tumor is tumor tissue or a portion thereof. In a moreparticular embodiment, said tumor tissue sample is a kidney tumor tissuesample from a patient whose response to anti-angiogenic treatment ofkidney cancer is to be predicted, or a breast tumor tissue sample from apatient whose response to anti-angiogenic treatment of breast cancer isto be predicted, or a colorectal tumor tissue sample from a patientwhose response to anti-angiogenic treatment of colorectal cancer is tobe predicted. Said sample can be obtained by conventional methods, e.g.,biopsy, surgical excision or aspiration, by using methods well known tothose of ordinary skill in the related medical arts and previouslydescribed in the context of the diagnostic method of the invention. Inone embodiment, the sample containing tumor cells is a sample of theprimary tumor. In another embodiment, the tumor is a metastatic tumorand the sample wherein the PD-ECGF levels are determined is a samplefrom the metastasis.

In another embodiment, the sample wherein the expression level ofPD-ECGF is determined is a biofluid from the patient suffering fromcancer whose response to treatment is to be predicted. In a preferredembodiment, the biofluid is selected from blood, particularly peripheralblood, plasma or serum. Methods for the obtention of blood, serum, andplasma samples are known by the skilled person and have been describedpreviously in the context of the diagnostic method of the invention.

In a particular embodiment method of the invention for predicting theresponse to an anti-angiogenic treatment, the sample wherein PD-ECGFexpression levels are determined is a plasma sample or a tissue sample.In a particular embodiment, PD-ECGF expression levels are determined ina plasma sample or in a breast tumor tissue sample when response toanti-angiogenic treatment of breast cancer is to be predicted. In aparticular alternative embodiment, PD-ECGF expression levels aredetermined in a plasma sample or in a kidney tumor tissue sample whenresponse to anti-angiogenic treatment of renal cell carcinoma (RCC) isto be predicted. In another particular alternative embodiment, PD-ECFGexpression levels are determined in a plasma sample or in a colorectaltumor tissue sample when response to anti-angiogenic treatment ofcolorectal cancer is to be predicted.

As previously described, gene expression levels can be quantified bymeasuring the messenger RNA levels of the gene or of the protein encodedby said gene or of the protein encoded by said gene, i.e. PD-ECGFprotein or of variants thereof. PD-ECGF protein variants have beendescribed above and are incorporated herein.

Methods to determine expression levels based on mRNA levels and proteinlevels, particularly PD-ECGF mRNA levels and PD-ECGF protein levels,have been described previously in the context of the diagnostic methodof the invention and incorporated herein.

In a particular embodiment, PD-ECGF expression levels to be determinedin the prediction of response to anti-angiogenic treatment method of theinvention are determined as PD-ECGF protein levels. In a more particularembodiment, PD-ECGF protein levels are determined by ELISA, western blotor by immunohistochemistry.

In a particular embodiment, the subject suffering from cancer whoseprediction of response to treatment is to be determined by the method ofthe invention is undergoing an anti-angiogenic treatment, wherein saidanti-angiogenic treatment of cancer is based on at least oneanti-angiogenesis agent.

Anti-angiogenic agents and treatments according to the inventioninclude, without limitation anti-VEGF agents, including monoclonalantibodies such as bevacizumab (Avastin, a recombinant humanizedmonoclonal IgG1 antibody that binds to and inhibits the biologicalactivity of human VEGFA in in vitro and in vivo assay systems), antibodyderivatives such as ranibizumab (Lucentis), or antibody fragments suchas Fab IMC 1121 or F200 Fab or orally-available small molecules thatinhibit the tyrosine kinases stimulated by VEGF such as lapatinib(Tykerb), sunitinib (Sutent), sorafenib (Nexavar), axitinib, andpazopanib; anti-fibroblast growth factor (anti-FGF) agents, such assuramin and its derivatives, pentosanpolysulfate, cediranib, pazopanib,or BIM 1120); anti-EGF agents, such as cetuximab, gefitinib or erlotiniband anti-HGF agents, such as ARQ197, JNJ-38877605, PF-04217903, SGX523,NK4, or AMG102; and antiangiogenic polypeptides such as angiostatin,endostatin, anti-angiogenic anti-thrombin III or sFRP-4.

Further anti-angiogenic agents include Marimastat; AG3340; COL-3,BMS-275291, Thalidomide, Endostatin, SU5416, SU6668, EMD121974,2-methoxyoestradiol, carboxiamidotriazole, CMIOI (GBS toxin),pentosanpolysulphate, angiopoietin 2 (Regeneron), herbimycin A,PNU145156E, 16K prolactin fragment, Linomide, thalidomide,pentoxifylline, genistein, TNP470, endostatin, paclitaxel, accutin,angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, plateletfactor 4 or minocycline.

Further anti-angiogenic agents include anti-angiogenic polypeptides,denoting polypeptides capable of inhibiting angiogenesis and including,without limitation, angiostatin, endostatin, anti-angiogenicanti-thrombin III, sFRP-4 as described in WO2007115376, an anti-VEGFantibody such as anibizumab, bevacizumab (avastin), Fab IMC 1121 andF200 Fab.

Further anti-angiogenic agents include pegaptanib, sunitinib, pazopanib,sorafenib, vatalanib and aflibercept (VEGF-Trap).

Further anti-angiogenic agents include VEGFR2 blocking antibodies, suchas Ramucirumab (IMC-1121B) and DC101 (also known as anti-Flk-1 mAb).

In a particular embodiment, the anti-angiogenic agent is selected fromthe group comprising sunitinib, bevacizumab, and DC101.

In one embodiment, the antiangiogenic treatment is not a treatmentcomprising an anthracyclin antibiotic. In another embodiment, theantiangiogenic treatment is not a treatment comprising doxorubicin. Inanother embodiment, the antiangiogenic treatment is not a treatmentcomprising an interferon. In another embodiment, the antiangiogenictreatment is not a treatment comprising a type I interferon and/or atype II interferon. In another embodiment, the antiangiogenic treatmentis not a treatment comprising IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω. Inanother embodiment, the antiangiogenic treatment is not a treatmentcomprising IFN-γ. In another embodiment, the antiangiogenic treatment isan adjuvant treatment, i.e. after the surgical excision of the tumor. Inone embodiment, the antiangiogenic treatment is not an adjuvanttreatment comprising interferon. In another embodiment, theantiangiogenic treatment is not an adjuvant treatment comprising a typeI interferon and/or a type II interferon. In another embodiment, theantiangiogenic treatment is not an adjuvant treatment comprising IFN-α,IFN-β, IFN-ε, IFN-κ and IFN-ω. In another embodiment, the antiangiogenictreatment is not an adjuvant treatment comprising IFN-γ.

Assays to determine the antiangiogenic activity of a particular agentare described, without limitation, in WO 2003086299.

In a second step of the prediction of response method of the invention,the expression levels of PD-ECGF in the sample from a subject sufferingfrom cancer whose prediction to anti-angiogenic treatment is to bedetermined are compared to a reference value.

In the context of the method of the invention for the prediction ofresponse of cancer in a subject to the treatment with anti-angiogenictherapy, a suitable reference value can be the PD-ECGF expression leveldetermined in a sample from a subject having cancer or having had cancerwhich has shown a good response to treatment with anti-angiogenictherapy, said expression levels having been determined at the time thatthe patient was being treated. In another embodiment, the referencevalue is the PD-ECGF levels in a healthy patient, i.e, a patient whichhas not been diagnosed with the type of cancer for which a prediction ofthe response to therapy is desired.

Once this reference value is established, the level of PD-ECGF expressedin the sample can be compared with said reference value, and thus beassigned a level of “increased” or “decreased” expression. For example,an increase in expression levels above the reference value of at least1.1-fold, 1.5-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more compared withthe reference value is considered as “increased” expression level. Onthe other hand, a decrease in expression levels below the referencevalue of at least 0.9-fold, 0.75-fold, 0.2-fold, 0.1-fold, 0.05-fold,0.025-fold, 0.02-fold, 0.01-fold, 0.005-fold or even less compared withreference value is considered as “decreased” expression level.

Thus, if an increased expression level of PD-ECGF in the sample from asubject suffering from cancer whose prediction of response to treatmentis to be determined when compared to a reference value is observed, itis indicative of a poor response to treatment. Alternatively, if adecreased expression level of PD-ECGF in the sample from a subjectsuffering from cancer whose prediction of response to treatment is to bedetermined when compared to the reference value, it is indicative of agood response to treatment.

Uses of the Invention

In another aspect, the invention relates to the use of PD-ECGF in thediagnosis of cancer in a subject and/or in the prediction of theresponse of a cancer patient to an anti-angiogenic treatment. More inparticular, the invention relates to the use of PD-ECGF in the diagnosisof cancer in a subject and/or in the prediction of the response of acancer patient to an anti-angiogenic treatment, wherein saidanti-angiogenic treatment is not doxorubicin or interferon therapy.

The terms “diagnosis of cancer” and “prediction of the response of acancer patient to an anti-angiogenic treatment” have been defined aboveand are equally applicable to the uses according to the presentinvention. In a preferred embodiment, the cancer is selected from thegroup consisting of renal cell carcinoma (RCC), breast cancer andcolorectal cancer. In a preferred embodiment, the use according to theinvention comprises the use of the PD-ECGF polypeptide. In oneembodiment, PD-ECGF polypeptide is used by determining the levels usinga reagent which is capable of specifically binding to PD-ECGF, such asan antibody or an aptamer. In a preferred embodiment, the use accordingto the invention comprises the use of the polynucleotide encoding thePD-ECGF polypeptide. In one embodiment, the polynucleotide encoding thePD-ECGF polypeptide is used by determining the levels using a reagentwhich is capable of specifically binding to the polynucleotide encodingthe PD-ECGF, such as a specific primer set or a probe.

The invention is described below by the following examples, which mustbe considered as merely illustrative and in no case limiting of thescope of the present invention.

EXAMPLES Example 1. Identification of Tissue PD-ECGF Levels as a CancerMarker for Detection, Diagnosis and Monitoring of Cancer in Patients.Determination of PD-ECGF Protein Levels in Non-Cancerous or Tumor TissueSamples by Inmunohistochemistry

PD-ECGF expression was analyzed in tumors from patients byimmunodetection. A mouse monoclonal anti-thymidime phosphorylase [P-GF,44C] antibody was used (Abeam, ab 3151).

Results showed that:

-   -   In 12 non-cancerous tissues, PD-ECGF was not detected; therefore        there is a significant difference between the PD-ECGF levels of        non-cancerous tissue and tumor mass in cancer patients        (Mann-Whitney test, p<0.0001) (FIG. 1).    -   Protein expression of PD-ECGF was observed in 67 (>97%) tissue        samples out of 69 RCC patients and in 9 (56%) out of 16 in        tissues samples from breast cancer patients analyzed by        inmunohistochemistry.    -   PD-ECGF has different locations (membrane, cytoplasm and        nucleus), which allows evaluating the different degrees of        expression according to location, and    -   PD-ECGF shows different expression levels depending on the        intrinsic characteristics of each tumor.

When PD-ECGF basal (pre-treatment) expression levels were analyzed, itwas observed that the tumors showed different basal expression levels(FIGS. 2 and 3), probably depending on the intrinsic characteristics ofeach tumor.

Example 2. Identification of Plasma PD-ECGF Levels as a Cancer Markerfor Detection, Diagnosis and Monitoring of Cancer in Patients.Determination of PD-ECGF Protein Levels in Plasma Samples by ELISA

PD-ECGF expression was analyzed in plasma from healthy subjects andcancer patients by ELISA. A human thymidine phosphorylase, TP ELISA Kit[CSB-E10814h] was used.

Results showed that:

-   -   Plasma PD-ECGF levels were not detectable in healthy subjects        compared to cancer patients. Quantitatively, there is a        statistically significant difference between the PD-ECGF levels        of healthy subjects and plasma levels of PD-ECGF in cancer        patients (Mann-Whitney test, p=0.0013) (FIG. 5).    -   Protein expression of PD-ECGF was observed in 14 (>87%) plasma        samples out of 16 RCC patients, in 11 (69%) out of 16 in plasma        samples from breast cancer patients and in 52 (90%) out of 58 in        plasma samples from colorectal cancer patients analyzed by        ELISA.    -   PD-ECGF shows different expression levels depending on the        characteristics of each patient.    -   When PD-ECGF basal expression levels were analyzed, it was        observed that the patients showed different basal expression        levels (FIGS. 6, 7 and 8), probably depending on the intrinsic        characteristics of each patient.

Example 3. Identification of PD-ECGF as a Predictive Factor of Responseto Anti-Angiogenic Treatment by Immunohistochemistry and ELISA

PD-ECGF expression was analyzed in tumors from patients byimmunodetection. A mouse monoclonal anti-thymidime phosphorylase [P-GF,44C] antibody was used (Abcam, ab 3151).

Results showed that:

-   -   PD-ECGF was evaluated in RCC tissue samples from 63 patients;        there is a significant association between higher tissue levels        of PD-ECGF and higher risk of early progression in RCC patients        (Kaplan-Meier and Gehan-Breslow-Wilcoxon test, p=0.045) (FIG.        4).

On the other hand PD-ECGF expression was analyzed in plasma frompatients by ELISA. A human thymidine phosphorylase, TP ELISA Kit[CSB-E10814h] was used.

Results showed that:

-   -   PD-ECGF was evaluated in colorectal cancer plasma samples from        52 patients; there is a significant association between plasma        levels of PD-ECGF and risk of early progression represented by a        significant hazard ratio for PD-ECGF levels in disease        progression in colorectal cancer patients (posterior probability        of Hazard ratio was used to evaluate the data, and posterior        probability that the hazard ratio be higher than 1 was equal to        0.041, which shows association between tumor progression and        PD-ECGF levels) (FIG. 9).    -   PD-ECGF was evaluated in RCC plasma samples from 14 patients;        there is a significant association between higher plasma levels        of PD-ECGF and higher risk of early progression in RCC patients        (Kaplan-Meier and Log rank (Mantel-Cox) tests, p<0.07). (FIG.        10).        Thus, PD-ECGF expression has potential value in predicting        cancer progression in treated patients. These findings support        the use of PD-ECGF as a novel biomarker with predictive value of        response to treatment for RCC, breast cancer, colorectal cancer        and other types of cancer.

Example 4. PD-ECGF Expression Levels and Cancer Prognosis

To analyze the relation between PD-ECGF expression levels and theprognosis of cancer, the inventors generated Kaplan-Meier survivalcurves based on cancer patients with low or high expression of PD-ECGFby using cBioPortal for Cancer Genomics (www.cbioportal.org) (Fig. A-M).

The following protocol was followed to generate FIGS. 11-23: select“Query” on the home page of the website www.cbioportal.org, select“studies in cancer patients (*)” from Select Cancer Study. In the“Select Genomic Profiles”, only select “mRNA Expression z-Scores (RNASeq V2 RSEM). “Enter Gene set”, input “TYMP”, and then click “Submit”.Oncoprint gives the percentage of alteration (see Table 1 and Table 2below) and Click “Survival” tab, overall survival Kaplan-Meier Estimatewill appear.

TABLE 1 Percentage of PD-ECGF alteration in cancer cell lines PD-ECGFStudies in cancer cell lines (% of alteration) Cancer Cell LineEncyclopedia (Novartis/Broad, 12 Nature 2012) All Complete tumors (877samples) NCI-60 Cell Lines (NCI, Cancer Res. 2012) All 7 completesamples (60 samples)

TABLE 2 Percentage of PD-ECGF alteration in cancer patients PD-ECGFStudies in cancer patients % of alteration Colorectal Adenocarcinoma(TCGA, Provisional) All 14%  Complete Tumors (7 samples) Glioblastoma(TCGA, Nature 2008) All Complete 2% Tumors (91 samples) Glioblastoma(TCGA, Cell 2013)All Complete Tumors 3% (291 samples) StomachAdenocarcinoma (TCGA, Provisional) All 3% Complete Tumors (33 samples)Kidney Renal Clear Cell Carcinoma (TCGA, 4% Provisional)All CompleteTumors (413 samples) Kidney Renal Clear Cell Carcinoma (TCGA, Nature 5%2013) All Complete Tumors (392 samples) Kidney Renal Papillary CellCarcinoma (TCGA, 5% Provisional)All Complete Tumors (161 samples) LungAdenocarcinoma (TCGA, Provisional)All 6% Complete Tumors (230 samples)Prostate Adenocarcinoma (MSKCC, Cancer Cell 2010) 2% All Complete Tumors(85 samples) Prostate Adenocarcinoma (TCGA, Provisional) All 4% CompleteTumors (332 samples) Testicular Germ Cell Cancer (TCGA, Provisional) All4% Complete Tumors (149 samples) Thymoma (TCGA, Provisional) All Tumors5% (124 samples) Thyroid Carcinoma (TCGA, Provisional) All Complete 6%Tumors (397 samples) Papillary Thyroid Carcinoma (TCGA, Cell 2014) All6% Complete Tumors (388 samples)Results showed the following:

-   -   colorectal adenocarcinoma cases with PD-ECGF alteration have        worse disease free survival than cases without PD-ECGF        alteration (FIG. 11),    -   glioblastoma cases with PD-ECGF alteration have worse overall        survival than cases without PD-ECGF alteration (FIG. 12),    -   kidney renal clear cell carcinoma cases with PD-ECGF alteration        have worse overall survival than cases without PD-ECGF        alteration (FIG. 13),    -   kidney renal clear cell carcinoma cases with PD-ECGF alteration        have worse disease free survival than cases without PD-ECGF        alteration (FIG. 14),    -   kidney renal clear cell carcinoma cases with PD-ECGF alteration        have worse overall survival than cases without PD-ECGF        alteration (FIG. 15),    -   kidney renal papillary cell carcinoma cases with PD-ECGF        alteration have worse disease free survival than cases without        PD-ECGF alteration (FIG. 16),    -   lung adenocarcinoma cases with PD-ECGF alteration have worse        overall survival than cases without PD-ECGF alteration (FIG.        17),    -   lung adenocarcinoma cases with PD-ECGF alteration have worse        disease free survival than cases without PD-ECGF alteration        (FIG. 18),    -   prostate adenocarcinoma cases with PD-ECGF alteration have worse        disease free survival than cases without PD-ECGF alteration        (FIG. 19),    -   testicular germ cell cancer cases with PD-ECGF alteration have        worse overall survival than cases without PD-ECGF alteration        (FIG. 20),    -   thymoma cases with PD-ECGF alteration have worse overall        survival than cases without PD-ECGF alteration (FIG. 21),    -   thymoma cases with PD-ECGF alteration have worse disease free        survival than cases without PD-ECGF alteration (FIG. 22), and    -   papillary thyroid carcinoma cases with PD-ECGF alteration have        worse overall survival than cases without PD-ECGF alteration        (FIG. 23).

1. A method for the diagnosis of cancer in a subject that comprises (i)determining the levels of PD-ECGF in a sample from said subject and,(ii) comparing the levels obtained in (i) to a reference value, whereinif the levels of PD-ECGF in a sample from the subject are increased whencompared to a reference value, then the subject is diagnosed withcancer, and if the levels of PD-ECGF in a sample from the subject aredecreased when compared to a reference value, then the subject is notdiagnosed with cancer.
 2. A method for predicting the response of asubject suffering from cancer to an anti-angiogenic treatment, whereinsaid anti-angiogenic treatment is not doxorubicin or interferon therapy,that comprises (i) determining the levels of PD-ECGF in a sample fromsaid subject and, (ii) comparing the levels obtained in (i) to areference value, wherein if the levels of PD-ECGF in a sample from thesubject are increased when compared to a reference value, it isindicative of a bad response to the anti-angiogenic treatment, and ifthe levels of PD-ECGF in a sample from the subject are decreased whencompared to a reference value, it is indicative of a good response tothe anti-angiogenic treatment.
 3. A method according to claim 2, whereinresponse is measured as early progression of cancer.
 4. A methodaccording to claim 2, wherein the anti-angiogenic treatment is initiatedafter the determination of the levels of PD-ECGF.
 5. The methodaccording to claim 1, wherein the sample is a tissue sample or abiological fluid sample.
 6. The method according to claim 5 wherein thetissue sample is a sample from the tumor, or wherein the biologicalfluid sample is a plasma sample.
 7. The method of claim 6 wherein thetumor is a primary tumor or a metastasis.
 8. A method according to claim1, wherein the reference value is determined in sample from a healthysubject or in a sample from a subject not diagnosed with cancer.
 9. Themethod according to claim 1, wherein the levels of PD-ECGF determinedare PD-ECGF protein levels.
 10. The method according to claim 9, whereinPD-ECGF protein levels are determined by immunohistochemistry, by ELISA,or by western blot.
 11. The method according to claim 1, wherein thecancer is selected from the group consisting of renal cell carcinoma(RCC), breast cancer and colorectal cancer.
 12. (canceled) 13.(canceled)
 14. The method according to claim 2, wherein the sample is atissue sample or a biological fluid sample.
 15. The method according toclaim 14 wherein the tissue sample is a sample from the tumor, orwherein the biological fluid sample is a plasma sample.
 16. The methodof claim 15 wherein the tumor is the primary tumor or a metastasis. 17.The method according to claim 2, wherein the levels of PD-ECGFdetermined are PD-ECGF protein levels.
 18. The method according to claim17, wherein PD-ECGF protein levels are determined byimmunohistochemistry, by ELISA, or by western blot.
 19. The methodaccording to claim 2, wherein the cancer is selected from the groupconsisting of renal cell carcinoma (RCC), breast cancer and colorectalcancer.